Search

Insulin is a pleiotropic hormone that elicits its metabolic and mitogenic actions through numerous rapid and reversible protein phosphorylations. The temporal regulation of insulin's intracellular signaling cascade is highly complex and insufficiently understood. We conduct a time-resolved analysis of the global insulin-regulated phosphoproteome of differentiated human primary myotubes derived from satellite cells of healthy donors using high-resolution mass spectrometry. Identification and tracking of ~13,000 phosphopeptides over time reveal a highly complex and coordinated network of transient phosphorylation and dephosphorylation events that can be allocated to time-phased regulation of distinct and non-overlapping subcellular pathways. Advanced network analysis combining protein-protein-interaction (PPI) resources and investigation of donor variability in relative phosphosite occupancy over time identifies novel putative candidates in non-canonical insulin signaling and key regulatory nodes that are likely essential for signal propagation. Lastly, we find that insulin-regulated phosphorylation of the pre-catalytic spliceosome complex is associated with acute alternative splicing events in the transcriptome of human skeletal muscle. Our findings highlight the temporal relevance of protein phosphorylations and suggest that synchronized contributions of multiple signaling pathways form part of the circuitry for propagating information to insulin effector sites. The temporal regulation of intracellular insulin signaling is not well studied. Here the authors conducted a time-resolved analysis of the global insulin-regulated phosphoproteome in human muscle cells, revealing synchronized signaling pathways for propagating information to insulin effector sites. [ABSTRACT FROM AUTHOR]

Background and Purpose: Remote ischaemic preconditioning (rIPC) for cardioprotection is severely impaired in diabetes, and therapeutic options to restore it are lacking. The vascular endothelium plays a key role in rIPC. Given that the activity of endothelial nitric oxide synthase (eNOS) is inhibited by proline‐rich tyrosine kinase 2 (Pyk2), we hypothesized that pharmacological Pyk2 inhibition could restore eNOS activity and thus restore remote cardioprotection in diabetes. Experimental Approach: New Zealand obese (NZO) mice that demonstrated key features of diabetes were studied. The consequence of Pyk2 inhibition on endothelial function, rIPC and infarct size after myocardial infarction were evaluated. The impact of plasma from mice and humans with or without diabetes was assessed in isolated buffer perfused murine hearts and aortic rings. Key Results: Plasma from nondiabetic mice and humans, both subjected to rIPC, caused remote tissue protection. Similar to diabetic humans, NZO mice demonstrated endothelial dysfunction. NZO mice had reduced circulating nitrite levels, elevated arterial blood pressure and a larger infarct size after ischaemia and reperfusion than BL6 mice. Pyk2 increased the phosphorylation of eNOS at its inhibitory site (Tyr656), limiting its activity in diabetes. The cardioprotective effects of rIPC were abolished in diabetic NZO mice. Pharmacological Pyk2 inhibition restored endothelial function and rescued cardioprotective effects of rIPC. Conclusion and Implications: Endothelial function and remote tissue protection are impaired in diabetes. Pyk2 is a novel target for treating endothelial dysfunction and restoring cardioprotection through rIPC in diabetes. [ABSTRACT FROM AUTHOR]

Cellular heterogeneity is a well-accepted feature of tissues, and both transcriptional and metabolic diversity have been revealed by numerous approaches, including optical imaging. However, the high magnification objective lenses needed for high-resolution imaging provides information from only small layers of tissue, which can result in poor cell statistics. There is therefore an unmet need for an imaging modality that can provide detailed molecular and cellular insight within intact tissue samples in 3D. Using GFP-tagged GLUT4 as proof of concept, we present here a novel optical mesoscopy approach that allows precise measurement of the spatial location of GLUT4 within specific anatomical structures across the myocardium in ultrathick sections (5 mm x 5 mm x 3 mm) of intactmouse heart.We reveal distinct GLUT4 distribution patterns across cardiac walls and highlight specific changes in GLUT4 expression levels in response to high fat dietfeeding, and we identify sex-dependent differences in expression patterns. This method is applicable to any target that can be labelled for light microscopy, and to other complex tissues when organ structure needs to be considered simultaneously with cellular detail. [ABSTRACT FROM AUTHOR]

Aims: Exercise training induces white adipose tissue (WAT) beiging and improves glucose homeostasis and mitochondrial function in rodents. This could be relevant for type 2 diabetes in humans, but the effect of physical fitness on beiging of subcutaneous WAT (scWAT) remains unclear. This translational study investigates if beiging of scWAT associates with physical fitness in healthy humans and recent‐onset type 2 diabetes and if a voluntary running wheel intervention is sufficient to induce beiging in mice. Materials and Methods: Gene expression levels of established beiging markers were measured in scWAT biopsies of humans with (n = 28) or without type 2 diabetes (n = 28), stratified by spiroergometry into low (L‐FIT; n = 14 each) and high physical fitness (H‐FIT; n = 14 each). High‐fat diet‐fed FVB/N mice underwent voluntary wheel running, treadmill training or no training (n = 8 each group). Following the training intervention, mitochondrial respiration and content of scWAT were assessed by high‐resolution respirometry and citrate synthase activity, respectively. Results: Secreted CD137 antigen (Tnfrsf9/Cd137) expression was three‐fold higher in glucose‐tolerant H‐FIT than in L‐FIT, but not different between H‐FIT and L‐FIT with type 2 diabetes. In mice, both training modalities increased Cd137 expression and enhanced mitochondrial content without changing respiration in scWAT. Treadmill but not voluntary wheel running led to improved whole‐body insulin sensitivity. Conclusions: Higher physical fitness and different exercise interventions associated with higher gene expression levels of the beiging marker CD137 in healthy humans and mice on a high‐fat diet. Humans with recent‐onset type 2 diabetes show an impaired adipose tissue‐specific response to physical activity. [ABSTRACT FROM AUTHOR]